Neuronal nicotinic receptors (nAChRs) .are involved in a range of cerebral functions including attention, learning, and memory. A loss of nAChRs subunits is associated with diseases such as Alzheimer's disease, dementia with Lewis bodies, Parkinson's disease and schizophrenia. Chronic nicotine exposure in tobacco smokers produces an increase in brain binding sites for nicotine. This increase in the number of nAChRs is commonly known as "upregulation". Specifically, chronic nicotine administration induces the up-regulation of mammalian a4(32 neuronal nicotinic acetylcholine receptor (nAChR) in the central nervous system (CNS). Recently we found that chronic exposure to a combined treatment with HIV-gpl2p, and galantamine produce a remarkable upregulation (11-fold) of the a7 nAChR in human macrophages in vitro. The goal of the proposed research is to gain insight into the molecular mechanisms of upregulation of 2 neuronal nicotinic receptors types;the oc4p2 and a7 nAChRs. Their upregulation is extremely relevant to nicotine tolerance, and the upregulation of the cc7 nAChR could have serious implications in processes ranging from neurodegeneration to inflammation in MTV-infected patients. The study of 2 different nAChRs will advance understanding of the molecular mechanisms governing upregulation of these receptors from a structural and functional perspective. The objective of the project is to define the molecular mechanisms involved in nicotine-induced upregulation a4|32 and cc7 nAChRs through multidisciplinary studies. In the project, we will use voltage-clamp whole-cell current electrophysiological recording to assess nAChR function, radioligand binding assays to ascertain numbers of a4p2-nAChR in total cell membranes and on the cell surface, site-directed mutagenesis approaches, and confocal microscopy to study the upregulation of the cc4p2 and a7 nAChRs. The Specific Aims are to: (1) define the structural and functional basis for the upregulation of the a4(32 nAChR induced by chronic nicotine exposure, (2) develop methods to incorporate fluorescent amino acids into the nAChR subunits using nonsense suppressor techniques that will allow the study nAChRs trafficking in vivo, (3) gain insight into the molecular basis for the upregulation of the cc7 nACnR induce by a comoined treatment with HTV-1 gpl20, nicotine and galantamine in vitro, (4) examine the functional state of the <x7 nAChR after chronic exposure to gp!20 in macrophages from HTV-infected patients and (5) express and purify the a7 nAChR extracellular domain for crystallization trials. Of biological significance, the project will uncover mechanisms involved in responses to chronic nicotinic ligand exposure that are relevant to nicotine tolerance, Alzheimer's disease, Parkinson's disease, schizophrenia and HTV-associated dementia. During the second phase of the present SNRP grant this research project on nAChRs will promote integration of basic, translational, and clinical research on ion channels.